1. Field of the Invention
This invention relates to a novel chorismate mutase (cm) gene, cloned from the potato cyst nematode Globodera rostochiensis, sequence polymorphisms in the Globodera cm genes, species-specific PCR (polymerase chain reaction) assays for specifically identifying G. rostochiensis and differentiating G. rostochiensis from G. pallida, and a strategy for developing G. rostochiensis-resistant potato cultivars.
2. Description of the Relevant Art
Potato cyst nematodes, G. rostochiensis and G. pallida, are internationally-recognized quarantine pests and considered the most devastating pests of potatoes due to annual worldwide yield losses estimated at 12.2%. First seen in the United States in 1941 on Long Island, G. rostochiensis was kept in check first with pesticides and later by crop rotation and planting of nematode-resistant potato varieties. These strategies had helped confine the pest to nine New York counties. Potato cyst nematodes continue to spread throughout North America and were recently detected in Idaho (G. pallida) and Quebec and Alberta, Canada (G. rostochiensis) creating serious problems for the potato industry, increasing the risk for the spread of these nematodes into other potato producing states, and resulting in a reevaluation of current quarantine practices.
G. rostochiensis is a sedentary endoparasitic nematode that has evolved an intimate parasitic relationship within host plant roots by transforming selected root cells into elaborate feeding structures that provide the nutrients required for the development and reproduction of the nematode. This root-infesting pest is nearly impossible to rid from contaminated soil because its eggs can lie dormant, protected within the dead body of its parent, for up to 30 years.
The endemic pathotype of G. rostochiensis, Ro1, continues to spread within New York state and a new pathotype, Ro2, that is virulent on potato cultivars resistant to Ro1, has become established in the field. Previously, more than 30 potato varieties that resist the original nematode race, Ro1, had been introduced. The key to Ro1 resistance is a potato gene called H1; however, H1 has no effect on the new nematode pathotype Ro2. To date, there are no commercially-available potato cultivars resistant to Ro2. If Ro2 were to become established in potato, tomato, and eggplant fields, it would cause significant annual losses.
Determining the G. rostochiensis pathotype with a traditional bioassay takes almost two years, during which time potato growers cannot determine if their fields contain Ro2. If Ro2 is found, the farmer is forced to abandon potato production or grow a non-profitable European variety, Sante. Thus, for eradication and quarantine purposes, there is a need for an assay to identify the G. rostochiensis pathotypes more quickly.
PCR-based assays have been described for detection and speciation of Globodera. The Random Amplified Polymorphic DNA (RAPD) method was used to determine inter- and intra-specific variation between populations of Globodera rostochiensis and G. pallida (Folkertsma et al. 1994. Phytopathology 84: 807-811). Two other published PCR methods for discrimination of potato cyst nematode species used a multiplex PCR with three primers based on the potato cyst nematode ribosomal internal transcribed spacer (ITS) region sequences: species-specific primers for G. rostochiensis and G. pallida in combination with a common primer which amplifies both (Mulholland et al. 1996. BCPC Symp. Proc. 65: 247-252; Bulman and Marshall. 1997. NZ J. Crop Hortic. Sci. 25: 123-129). The development of genotypic methods with the ability to precisely discriminate among the different species of Globodera is essential for effective monitoring and surveillance to determine the prevalence of these organisms in the environment, to facilitate implementing specific disease control strategies, and for accurately selecting areas for quarantine. There still exists a need for specific primers and methods capable of specifically identifying and differentiating pathogenic Globodera species. Nematode parasitism genes play important roles in nematode infection and parasitism of host plants and they have been suggested to have evolved much more quickly than ribosomal DNA; therefore, the parasitism gene, cm, isolated from the nematode is expected to be more suitable for the development of diagnostic methods/markers that distinguish Globodera species within a genus.
Plant-mediated RNA interference (RNAi) has been used to target nematode parasitism genes and helped attain broad resistance against four root-knot nematode species in the model plant Arabidopsis (Huang et al. 2006. Proc. Natl. Acad. Sci. USA 103: 14302-14306). The double-stranded (dsRNA) or small interfering (siRNA) molecules were taken up by the nematode from soaking solution (in vitro) or from plant tissue (in planta). RNAi has been observed to function in both cyst and root-knot nematode species (Lilley et al. 2007. Molecular Plant Path. 8: 701-711). Production of parasite-specific dsRNA in plant cells has been suggested as a novel and durable strategy for control of plant parasitic nematodes including cyst nematodes (e.g. Gheysen and Vanholme. 2007. Trends in Biotech. 25: 89-92; Steeves et al. 2006. Func. Plant Biol. 33: 991-999).
The use of nematode resistant cultivars is the most economical and environmentally-safe means of nematode control; therefore, there is also a need for Ro2-resistant cultivars.